ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs) and inactivation by GTPase-activating proteins (GAPs). Two general types of GEPs that have been involved in most of these studies are ~200-kDa proteins that are inhibited by BFA (a drug that reversibly interferes with protein secretion and causes disintegration of Golgi cisternae) and smaller ~50-kDa GEPs that are BFA-resistant. All GEPs have so-called Sec7 domains of ~200 amino acids that are responsible for the GEP activity, as well as its BFA sensitivity. The group had earlier purified, from bovine brain, two BFA-inhibited GEPs (BIG1 and BIG2) that behaved on gel filtration as molecules of ~670 kDa. Immunoprecipitation with specific, affinity-purified anti-peptide antibodies demonstrated that they are to a large extent components of the same macromolecular complexes. The 50-kDa cytohesin-1, cloned by its interaction with integrin, had been shown by the group to be a BFA-insensitive ARF GEP. Amino acid sequences for cytohesin-2 predicted by independent cDNA clones differ only in the presence or absence of a single glycine. The group demonstrated the existence of two mRNA isoforms that differ in the same way for each of three known cytohesins and identified a new cytohesin-4 with only one mRNA. To elucidate mechanism(s) that control the single glycine insertion, genes for cytohesin-1 and -4 were compared, revealing remarkable similarity of structure except for an extra 3-bp exon in cytohesin-1. The extra glycine alters PH domain structure resulting in significant differences in cytohesin interaction with specific phosphoinositides. Mechanisms that regulate the alternative splicing of cytohesins 1, 2, and 3, as well as the cell-specific expression of individual cytohesins are being investigated. In other studies, a novel ARF GEP, unlike any already known, was cloned and characterized. The ~100-kDa protein contains the Sec7 domain found in all ARF GEPs, but shares no other recognized domain structures. Its activity is not affected by brefeldin A or phospholipids, and it appears to function preferentiallly as a GEP for ARF6. The mRNA is particularly abundant in peripheral blood leukocytes, brain, and spleen. Immunoreactive endogenous ARF 6 and the new GEP100 were partially co-localized in vesicular structures and the GEP immunofluorescence also coincided with that of EEA-1, a marker for early endosomes. ADP-ribosylation factor domain protein 1 (ARD1), initially cloned in the laboratory, differs from other ARFs by the presence of a 46-kDa amino-terminal extension (p5), which acts as a GTPase-activating protein (GAP) for its ARF domain (p3). Similar to ARF GAPs, the GAP domain of ARD1 contains a zinc finger motif and arginine residues that are critical for activity. It differs from other ARF GAPs in its covalent association with the GTP-binding domain and the specificity of its GAP activity for the ARF domain of ARD1. ARFs are presumed to play a key role in the formation of intracellular transport vesicles and in their movement from one compartment to another. Both overexpressed and endogenous ARD1 were associated with Golgi and lysosomal membranes, consistent with a role in the formation or function of lysosomes and in protein trafficking bettween Golgi and lysosomes. Interaction of ARD1 and cytohesin-1 was found in a yeast two-hybrid screen. Cytohesin-2 failed to interact in this system and had much less GEP activity toward ARD1 than did cytohesin-1, although they were equally active with ARF1. Preferential physical interaction was also shown in vitro and residues responsible for specificity of the cytohesin-1/ARD1 interaction were identified. ARD1 "knock-out" mice were finally obtained this year and new clues to the physiological function of this protein are emerging.